Magnetic orientational linear dichroism (MOLD) spectroscopy was found to be useful for detecting the initiation of the agglomeration process of COOH-modified iron-oxide magnetic nanoparticles (MNPs) in water, which was produced by the addition of a small amount of a cationic surfactant or a metal ion. The critical association of MNPs leading to agglomeration was detected from the increase of the maximum MOLD value as well as from the shift of the maximum wavelength of the MOLD spectrum. The magnetic field dependence of MOLD was analyzed by the Langevin equation, and the apparent association numbers of MNPs in the agglomerates were obtained.